Questions:
1. How far upstream genes (30-100bp more) do I need to include? Do I even need to include them at all, since I already have t7 promoter?
2. How important to know the GC content of the amplicons? Would it matter or not?

Questions:1. How far upstream genes (30-100bp more) do I need to include? Do I even need to include them at all, since I already have t7 promoter?2. How important to know the GC content of the amplicons? Would it matter or not?

Thank you for your time.

For upstream regions, you only need to include it if you want to express the operon from the native promoter. If that's the case you want to include the promoter, which is usually within 100-150 bases upstream of the start IN ADDITION TO any potential transcriptional regulatory regions upstream of the promoter. When I do these types of amplifications, I try to get at least 250-400 bp upstream of the start to have a high probability of including any transcriptional regulatory regions for the gene/operon.

On the other hand, if you're looking to induce expression in your E. coli strain, you don't need any of the native promoter, just clone it in downstream of your T7 (or whatever other promoter is in the vector) and induce expression.

The GC content may affect amplification. If there's high AT, the amplification of a 5 kb piece could be troublesome, as could too high GC. The GC content shouldn't affect expression too much, though.

One question, though... why are you growing your E. coli at 30 or 25 C?

For upstream regions, you only need to include it if you want to express the operon from the native promoter. If that's the case you want to include the promoter, which is usually within 100-150 bases upstream of the start IN ADDITION TO any potential transcriptional regulatory regions upstream of the promoter. When I do these types of amplifications, I try to get at least 250-400 bp upstream of the start to have a high probability of including any transcriptional regulatory regions for the gene/operon.

On the other hand, if you're looking to induce expression in your E. coli strain, you don't need any of the native promoter, just clone it in downstream of your T7 (or whatever other promoter is in the vector) and induce expression.

The GC content may affect amplification. If there's high AT, the amplification of a 5 kb piece could be troublesome, as could too high GC. The GC content shouldn't affect expression too much, though.

One question, though... why are you growing your E. coli at 30 or 25 C?

Thank you for your explanation.

I possibly will grow my E. coli at that temperature because one of the protein is a possible membrane-bound and I would like to increase the solubility of the protein (Is this correct?). The operon is from a gram negative bacterium that lives at 20C so I would like to copy the native environment as much as possible. I'm not sure if I'm doing this correctly but any input will be appreciated. Thanks again.

I possibly will grow my E. coli at that temperature because one of the protein is a possible membrane-bound and I would like to increase the solubility of the protein (Is this correct?). The operon is from a gram negative bacterium that lives at 20C so I would like to copy the native environment as much as possible. I'm not sure if I'm doing this correctly but any input will be appreciated. Thanks again.

Well, mimicking the environment of your bacterium for expression of the operon in E. coli kinda goes out the window, because E. coli is a completely different bug than yours (especially if yours is growing at 20 C). For looking at expression of your operon in E. coli in certain conditions, you assume E. coli has similar signalling events that occur in response to similar environments, resulting in expression of the genes in question. That's a big assumption, unless prior work has been done showing that those things are equal between E. coli and your bacterium.

As for growing at a lower temperature to increase solubility, I cannot speak to that.

Expressing your operon in E. coli allows you to identify the proteins being produced, and possibly to purify them for study. Studying the expression of the proteins in response to certain conditions, however, needs to be done in your particular organism, in my opinion. But that will depend on what exactly you're attempting to do. If you're looking to see if a protein is a membrane protein and seeing if E. coli inserts it into its membrane, then that can be done, but other things like looking at how certain conditions affect expression or function of an operon, that needs to be done in your particular organism, not E. coli.

I don't have enough information to go into specifics about whether or not your methods are correct for the type of study you are planning.

Well, mimicking the environment of your bacterium for expression of the operon in E. coli kinda goes out the window, because E. coli is a completely different bug than yours (especially if yours is growing at 20 C). For looking at expression of your operon in E. coli in certain conditions, you assume E. coli has similar signalling events that occur in response to similar environments, resulting in expression of the genes in question. That's a big assumption, unless prior work has been done showing that those things are equal between E. coli and your bacterium.

As for growing at a lower temperature to increase solubility, I cannot speak to that.

Expressing your operon in E. coli allows you to identify the proteins being produced, and possibly to purify them for study. Studying the expression of the proteins in response to certain conditions, however, needs to be done in your particular organism, in my opinion. But that will depend on what exactly you're attempting to do. If you're looking to see if a protein is a membrane protein and seeing if E. coli inserts it into its membrane, then that can be done, but other things like looking at how certain conditions affect expression or function of an operon, that needs to be done in your particular organism, not E. coli.

I don't have enough information to go into specifics about whether or not your methods are correct for the type of study you are planning.

Thank you for your response.

I actually have expressed one of the protein (His-tagged and untagged) in E. coli and did a specific enzymatic assay with the E. coli crude extracts. Both of the crude extracts showed activity.

In addition to that, I would like to express the other proteins in the operon because I think they might need each other and maybe trying to find out what others may do.

I actually have expressed one of the protein (His-tagged and untagged) in E. coli and did a specific enzymatic assay with the E. coli crude extracts. Both of the crude extracts showed activity.

In addition to that, I would like to express the other proteins in the operon because I think they might need each other and maybe trying to find out what others may do.

I see. That sort of analysis is fine in E. coli. I will caution though that there could be other interactions in your organism involved in the activity of these proteins, and expression in E. coli may not replicate those interactions. But you have to start somewhere, and showing an interaction between the proteins in E. coli will provide some of that information.

Is there any reason you cannot do these analyses in your particular organism?

I see. That sort of analysis is fine in E. coli. I will caution though that there could be other interactions in your organism involved in the activity of these proteins, and expression in E. coli may not replicate those interactions. But you have to start somewhere, and showing an interaction between the proteins in E. coli will provide some of that information.

Is there any reason you cannot do these analyses in your particular organism?

I believe one from the team is studying RNA which leaves me with the protein There are many reasons that I can't do analyses in the organism itself and one of them is it is a slow-growing bacterium. It'll take me weeks before I can play with it. But, I'll do that alongside with E. coli. So in the meantime, I'll just clone a possible operon and play with it. Maybe it will do something, maybe it won't.